Generation of mouse hippocampal brain organoids from primary embryonic neural stem cells

Here we present a protocol to generate standardized cerebral organoids with hippocampal regional specification using morphogen WNT3a. We describe steps for isolating mouse embryonic (E14.5) neural stem cells from the brain subgranular zone, preparing organoids samples for immunofluorescence, calcium imaging, and metabolic profiling. This protocol can be used to generate mouse brain organoids for developmental studies, modeling disease, and drug screening. Organoids can be obtained in one month, thus providing a rapid tool for high-throughput data validation. For complete details on the use and execution of this protocol, please refer to Ciarpella et al. "Murine cerebral organoids develop network of functional neurons and hippocampal brain region identity".¹.